[unreadable] The theoretical basis of gene therapy is the correction of a genetic defect through the transduction of a functional gene into the tissues of the recipient. A variety of technologies now exist for the increasingly efficient introduction of genetic material into a gene therapy recipient. However, rejection of the transduced gene by the recipient immune system remains a persistent obstacle to stable long-term expression. In this grant, we propose a strategy that circumvents the obstacle of immune rejection by utilizing a veto cell based approach to selectively induce immunological tolerance to the therapeutic gene. CDS* veto cells function as deletional antigen presenting cells, which eliminate T cells that recognize antigens presented by the MHC class I of the CDS* veto cell. Thus, to engineer a recipient veto cell that will delete T cells capable of recognizing the product of a therapeutic gene, one must both generate CD8+ veto cells from the recipient and insert the therapeutic gene into the CDS* veto cells. The central hypothesis of this grant application is that recipient derived CDS* veto cells, which are transduced with the therapeutic gene, can be used to selectively tolerize the recipient immune system to the therapeutic gene. In this way, immune based rejection of the transduced gene will be eliminated as an obstacle to gene therapy. Specific Aim 1- Generation of recipient derived CDS* veto cells expressing a transduced gene. In this aim, we will optimize the conditions required to rapidly and efficiently isolate large numbers of gene transduced CD8+ veto cells from recipient tissues. To perform as sophisticated an analysis as possible, we will utilize a model transgene for which a large arsenal of tools exist to analyze immune responses (chicken ovalbumin (OVA)). This optimization will include maximizing expansion of CDS* veto cells to optimize yields and maximizing the efficiency of gene transduction, expression and presentation on MHC class I molecules. Specific Aim 2- Assess the ability of gene therapy transduced CDS* veto cells to induce antigenspecific tolerance to the therapeutic gene. In this aim, we will test the ability of the recipient derived OVA expressing CDS* veto cells to tolerize anti- OVA T cell responses. This will be accomplished by both studying OVA specific veto activity in vitro and by performing in vivo tolerization experiments. By combining the more reductionist in vitro systems with the more physiological in vivo systems, we will be able to assess both mechanistic determinations and physiological efficacy. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]